The present invention relates to a method of and apparatus for measuring spectral absorption in scattering objects, e.g., suspensions, powder, etc., and, more particularly, to a method of and apparatus for measuring spectral absorption characteristics of a component transmitted or reflected in a specific direction when a beam is applied to a specimen from a specific direction.
The present invention also relates to a method of and apparatus for measuring a microscopic absorption distribution of opaque specimens, e.g., biological specimens, and, more particularly, to a method of and apparatus for measuring a microscopic absorption distribution, wherein unnecessary scattered light is removed to improve the resolution so that it is possible to accurately measure absorption in a very small region of a specimen.
Since the discovery of X-rays, techniques of externally observing the inside of a living body (e.g., human body) without damaging it (i.e., a bloodless or non-destructive measuring method) have been strongly demanded and developed in the field of biology, particularly in the medical field. These techniques employ gamma rays and X-rays, which have the shortest wavelengths among the electromagnetic waves, and radio waves, which have the longest wavelengths among them. The technique that employs the former has already been put to practical use as X-ray CT, and the technique that employs the latter as NMR-CT (Magnetic Resonance Imaging, i.e., MRI).
On the other hand, fewer attempts have been made to apply spectroscopy that deals with the measurement and analysis of ultraviolet, visible, near infrared and infrared spectra, which is widely employed in the fields of physics and chemistry, to in vivo measurement. This is because biometry that employs light, particularly the one that utilizes the process of absorption or emission of light has many problems left unsolved in terms of "quantitativeness", which is the most basic matter. This is the reason why reproducibility is inferior and reliability is low in regard to the absolute values obtained in measurement that is conducted at the present time by using, for example, an apparatus that measures reflected spectra with a solid-state device, or a highly sensitive TV camera.
In a case where light is applied to a scattering object such as an organic tissue, if the light is received face to face at 180.degree., it is possible to take out rectilinearly propagating light to some extent. However, the spatial resolving power is not very high in the present state of art.
The difference in the spatial resolving power between X-rays and light cannot be made up in the present state of art. However, employment of light rays, particularly near infrared rays will enable imaging of a tissue oxygen concentration from the hemoglobin in the blood. These light rays will give information which is different from that obtained by other techniques such as NMR-CT and X-ray CT.
As for relatively thin tissues with a thickness of 3 to 5 cm, it is possible to detect light transmitted thereby. This means that "photo-roentgenography" can be used for diagnostic purposes. The women's breasts have relatively homogeneous tissues and hence readily transmit light, and it is easy to detect the light transmitted thereby (thickness: up to about 3 cm) owing to the configuration. For this reason, "photo-roentgenography" has been employed for a medical examination for breast cancer for a long time under the name of "diaphanography (lightscanning)".
Under these circumstances, the present inventor disclosed that a plane wave mixed in scattered light can be separated therefrom for observation by detecting only the 0-order spectrum (corresponding to the first dark ring of an Airy's disk) of the Fraunhofer diffraction image (Airy's disk) of the plane wave and, by so doing, most of the scattered light can be removed. See, for example, Japanese Patent Application Nos. 01-62898 (1989), 01-250034 (1989) and 02-77690 (1990). More specifically, when only a 0-order Fraunhofer diffraction pattern of a plane wave as signal light is to be detected, the degree of separation of incoherent scattered light from the plane wave is given by
(scattering intensity)/(transmitted plane wave intensity).apprxeq.(.lambda./Dr).sup.2
In other words, the larger the beam diameter or the entrance aperture diameter Dr of a highly directional detecting system, e.g., a heterodyne light-receiving system, a Michelson light-receiving system, a highly directional optical system, etc., in comparison to the wavelength .lambda., the more the scattered light attenuates, and the more the scattered light can be separated from the plane wave. As one example of a highly directional optical system used to realize such observation, the present inventor proposed an optical system comprising two pinholes P.sub.1 and P.sub.2 which are spaced apart from each other, as shown in FIG. 27. This optical system is arranged such that 0-order light is detected by a detector 23 through the pinhole P.sub.2. The present inventor also proposed a highly directional optical system comprising a hollow, straight, long and thin glass fiber 35 the inner wall surface of which is coated with a light absorbing material, e.g., carbon, as shown in FIG. 28. Further, the present inventor proposed various highly directional optical systems such as those shown in FIGS. 29 to 36: a highly directional optical system (FIG. 29) comprising an objective lens Ob and a pinhole P that is disposed on the focal plane thereof to pass only a 0-order Fraunhofer diffraction pattern formed by the objective lens Ob; a highly directional optical system (FIG. 30) comprising a graded-index lens GL and a pinhole P (similar to that shown in FIG. 29) that is disposed on the focal plane at one end of the graded-index lens GL; a highly directional optical system (FIGS. 31 and 32) in which the pinhole P is replaced with an optical fiber SM that functions in the same way as the pinhole P; a highly directional optical system (FIGS. 32 and 35) in which an objective lens Ob2 which is similar to an objective lens Ob1 at the entrance side is disposed at the exit side of the pinhole P or the optical fiber SM in the above-described highly directional optical systems; and a highly directional optical system (FIGS. 34 and 36) in which a graded-index lens GL2 which is similar to a graded-index lens GL1 at the entrance side is disposed at the exit side of the pinhole P or the optical fiber SM.
Incidentally, there are known methods of measuring absorption in opaque specimens, for example, the opal glass method in which a rectilinear component and a transmission and scattering component of a specimen that causes scattering are uniformly scattered by use of opal glass to measure a transmission integral extinction of the specimen [see, for example, Kazuo Shibata "Photobiology Series: Introduction to Spectral Measurement", pp.62-82 (Jun. 20, 1976, Kyoritsu Shuppan K. K.)]. Heterogeneous systems such as suspensions of particles, for example, cells, granules, solid powder, etc. absorb and scatter light, in general. Accordingly, it is difficult to obtain only absorption wavelength characteristics of such heterogeneous systems. For this reason, it is conventional practice to obtain a quantity with which real absorption wavelength characteristics can be approximated. More specifically, a transmission integral extinction is obtained to replace absorption. The transmission integral extinction is the cologarithm of the ratio of a bundle of light rays attenuated by both absorption and scattering to the incident light rays, which is not coincident with absorption characteristics, in general. In order to enable the transmission integral extinction to be approximated to real absorption characteristics as much as possible, if parallel transmitted rays and scatteringly transmitted rays are detected at the same capturing rate by a detector, the effect of scattering on the ratio of the light rays becomes small. As a method for this purpose, the opal glass method has been put to practical use. In addition, the transmission integrating sphere method, photoelectric surface contact method, etc. have been put to practical use as methods of minimizing the effect of scattering by capturing the entire transmitted rays comprising parallel transmitted rays and scatteringly transmitted rays. A method that utilizes both the contact and scattering methods jointly has also been employed as an intermediate method between the detection of parallel transmitted light rays and scatteringly transmitted light rays at the same capturing rate and the detection of the entire transmitted light rays.
Meantime, a measuring method such as that shown in FIG. 37 has heretofore been employed to measure a microscopic absorption distribution in a specimen that causes scattering. More specifically, light from a light source that emits light over a wide spectral range is passed through an interferometer for Fourier spectroscopy and then sent to either a transmission optical path or a reflection optical path through a transmission/reflection switching mirror. If the transmission optical path is selected, the illuminating light is condensed to a very small point on a specimen placed on a specimen table through a lower Cassegrain system that functions as a condenser lens. Light that is transmitted through the measuring point and light that is forwardly scattered at the measuring point are focused on an aperture through an upper Cassegrain system that functions as an objective lens, and the light that passes through the aperture is made incident on a detector to measure absorption characteristics at the measuring point. Thus, it is possible to measure a transmission microscopic absorption distribution in the specimen by repeating measurement similar to the above with the specimen table being scanned in directions X and Y. If the switching mirror is changed over to the reflection optical path, the illuminating light is condensed to a point on the specimen through the upper Cassegrain system, and light that is reflected and scattered backwardly from the measuring point is focused on the aperture through the same upper Cassegrain system. Thus, a reflection microscopic absorption distribution in the specimen can be measured in the same way as the above.
FIG. 38 shows another conventional microspectroscopic method that employs a combination of an optical microscope and a spectrophotometer to observe an absorption spectrum of a very small region. Light from a light source l is formed into monochromatic light through a spectroscope m.sub.0 to illuminate a diaphragm (pinhole) p. With the diaphragm p defined as a light source of a microscope system, light is passed through an illuminating microscope m.sub.1. In consequence, a reduced image of the diaphragm p is formed on a specimen plane s. This image is enlarged through another microscope m.sub.2 and led to a detector d. If a specimen is placed at the position s where the reduced image of the diaphragm is formed, it is possible to measure absorbance of only a local region in the specimen.
Incidentally, if the same measuring method that is used for a specimen that causes no scattering is employed to obtain an absorption spectrum of a specimen that causes scattering, the effect of scattering is large, so that it is impossible to obtain an accurate absorption spectrum. As techniques of measuring absorption in opaque specimens, methods wherein a transmission integral extinction is measured by use of opal glass, an integrating sphere, etc., are known, as described above.
The above-described conventional methods, however, suffer from problems stated below: (1) the opal glass method involves the disadvantage that the light scattering power undesirably changes in accordance with wavelength; (2) the transmission integrating sphere method involves the disadvantage that a white reflecting material in the integrating sphere greatly decreases in reflectivity at a short wavelength, particularly near the ultraviolet region, even in the case of MgO powder, which is known as the best reflecting material, so that no reliable data can be obtained; (3) the photoelectric surface contact method that employs two detectors involves difficulty in obtaining the same wavelength sensitivity characteristics, whereas the photoelectric surface contact method that employs a single detector involves difficulty in installing a specimen and a control within a limited space; and (4) the method that employs both the contact and scattering methods jointly suffers from the problem that it is necessary to properly select the size of a specimen and the distance and size of the detector, although it is superior to the former three methods.
In addition, the four conventional methods involve the common disadvantage that there are cases where a transmission integral extinction measured cannot be approximated to the absorption wavelength of suspended particles. More specifically, as the intensity of reflected rays increases, it becomes impossible to make approximation. When the scattering spatial pattern that is formed by a specimen depends upon wavelength, the wavelength change of scatteringly transmitted rays becomes different from that of the scatteringly reflected rays, so that no approximation can be made. In addition, it is difficult to perform absorption measurement that provides spatial resolving power such as specifies a location of absorption. Although the conventional methods are usable for measurement of absorption in sparse heterogeneous systems such as dilute suspensions, these methods cannot be applied to measurement of absorption in dense translucent objects such as biological specimens when scattering is so strong that the relationship of Kubelta-Munk is valid.
The microspectroscopic measuring method for infrared region that employs a Fourier spectroscope and the microspectroscopic measuring method for visible region that employs a diffraction grating spectroscope, which have been described above, have no measures taken to deal with opaque specimens that cause scattering and therefore involve large errors in the measurement of such specimens, so that no reliable data can be obtained. In other words, since unnecessary scattered light mixes in from the surroundings including the front and rear of the measuring point, it is impossible to measure accurate absorption characteristics. In addition, since Fraunhofer diffraction patterns other than the 0-order diffraction pattern enter the objective lens, the resolution is limited. The measuring method that is a simple combination of the measuring method employing opal glass or an integrating sphere, developed as a method of measuring absorption in opaque specimens, and the microspectroscopic measuring method involves the problem that the detected signal light is weak so that it is difficult to effect measurement, and it cannot therefore be put to practical use. Even if the detection sensitivy is improved markedly, since the method that employs opal glass or an integrating sphere is merely an approximation method, it cannot be used for a specimen for which approximation cannot be made when the intensity of reflected rays is high or when the wavelength change of scatteringly transmitted rays is the same as that of scattering reflected rays, due to large errors. Even if such a measuring method can be used, the spatial resolving power is deteriorated. Thus, there is no proper measuring method for an absorption spectrum in a very small region of an opaque specimen that causes scattering in the present state of art.